AAV-EPO for treating companion animals

ABSTRACT

Compositions and methods are provided for treating companion animals are provided. An adeno-associated viral vector is provided which includes a nucleic acid molecule comprising a sequence encoding erythropoietin (EPO). In desired embodiments, the subject is a cat or dog.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED IN ELECTRONIC FORM

Applicant hereby incorporates by reference the Sequence Listing materialfiled in electronic form herewith. This file is labeled“15-7472PCT_Seq_Listing.txt”.

BACKGROUND OF THE INVENTION

Erythropoietin (EPO) is a hormone made predominantly within theperitubular cells of the kidney. It acts on the bone marrow, stimulatingerythropoiesis. Erythropoietin also controls apoptosis (programmed celldeath) of mature red blood cells. Renal disease reduces erythropoietinproduction. In humans, the management of anemia in chronic kidneydisease has been revolutionized by the development of recombinant humanerythropoietin (epoetin). Many of the symptoms that had been ascribed tochronic kidney disease such as fatigue, lethargy, somnolence andshortness of breath, which all impact unfavorably on quality of life,were resolved or markedly improved when anemia was corrected.

There are over 2 million cats and 350,000 dogs that suffer from chronickidney disease (CKD). Companion animals with CKD-related renal failuresuffer in similar ways. They do not have sufficient EPO and subsequentlybecome very anemic. In the past veterinarians have given humanrecombinant EPO until the animals would develop an immune response tothe infused EPO. Effectively, this leaves no long term treatment in themarket for a very well understood physiological process that has a clearneed in the clinic.

Therefore, compositions useful for expressing EPO in subjects,particularly companion animals, are needed.

SUMMARY OF THE INVENTION

Novel engineered erythropoietin (EPO) constructs are provided herein.These constructs can be delivered to subjects in need thereof via anumber of routes, and particularly by expression in vivo mediated by arecombinant vector such as a recombinant adeno-associated virus (rAAV)vector.

In some embodiments, the EPO is encoded by an endogenous sequence. Thatis, the EPO sequence is derived from the same subject species for whichadministration is ultimately intended.

In some embodiments, a pharmaceutical composition comprising apharmaceutically acceptable carrier and a recombinant vector asdescribed herein is provided. Also provided are methods for treatingchronic kidney disease by administering to a subject in need thereof arecombinant vector described herein that has an expression cassette,wherein said expression cassette further comprises regulatory controlsequences which direct expression of the EPO construct in the subject.In some embodiments, the subject being treated is a companion animal. Inone embodiment, the subject is a feline. In another embodiment, thesubject is a canine. As used herein, the terms “patient” and “subject”are used interchangeably, and can refer to a human or veterinarysubject.

In yet another embodiment, methods for increasing the amount ofcirculating EPO in a subject comprising providing a recombinant vectordescribed herein that has an expression cassette encoding EPO.

The recombinant vectors described above can be used in a regimen fortreating chronic kidney disease and other conditions characterized by adecrease in the amount of circulating red blood cells.

Other aspects and advantages of the invention will be readily apparentfrom the following detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing hematocrits of cats treated with AAV8expressing feline erythropoietin. Dashed lines indicate the normalrange.

FIG. 2 is a graph showing hematocrits of dogs treated with AAV8expressing canine erythropoietin. Dashed lines indicate the normalrange.

FIG. 3A shows the canine EPO propeptide sequence, with the leadersequence underlined. FIG. 3B shows the feline EPO propeptide sequence,with the leader sequence underlined.

FIG. 4 is a graph showing hematocrits of cats treated with AAV8expressing feline erythropoietin. Cats were treated with 3.0×10⁷ GC,3.0×10⁸ GC, 3.0×10⁹ GC, or 3.0×10¹⁰ GC AAV8f.EPO.

DETAILED DESCRIPTION OF THE INVENTION

Adeno-associated viral vectors carrying EPO expression constructs havebeen developed for use in subjects including companion animals (e.g.,feline and canine). Though likely effective, a recombinant canine orfeline specific EPO protein therapeutic would cost much more to developand manufacture than a viral vector mediated system for delivery of EPOto the affected animal. With a viral vector therapeutic, there is alsothe convenience of being able to treat the animal once, as opposed tofrequent injections of recombinant EPO. Stable expression of EPO wouldcorrect anemia and give the animal an improved quality of life. The EPOconstructs described herein are also characterized in that they providean EPO sequence which is endogenous to the subject, which reduces therisk of the subject developing an immune response to a non-nativeprotein.

Also provided are uses for the constructs described herein. Delivery ofthese constructs to subjects in need thereof via a number of routes, andparticularly for expression in vivo which is mediated by a recombinantvector such as a rAAV vector, is described. In one embodiment, methodsof using the constructs in regimens for treating chronic kidney diseasein a subject in need thereof and increasing the EPO in a subject arealso provided. In one embodiment, methods of using these constructs inregimens for treating anemia in a subject in need thereof are provided.In another embodiment, the subject's anemia is related to the use ofother medications. Possible medications which contribute to anemiainclude, but are not limited to, HIV/AIDS treatments (including AZT) andcancer therapeutics, including chemotherapy. In another embodiment, thesubject's anemia is related to a medical condition. Possible medicalconditions that contribute to anemia include, but are not limited to,cancer, HIV/AIDS, rheumatoid arthritis, Crohn's disease and otherchronic inflammatory diseases and dysfunctional bone marrow (e.g.,aplastic anemia, leukemia, myelodysplasia or myelofibrosis), multiplemyeloma, myeloproliferative disorders and lymphoma, hemolytic anemia,sickle cell anemia and thalassemia. In addition, methods are providedfor enhancing the activity of EPO in a subject.

EPO is expressed in vivo as a propeptide, with the leader sequencessharing some homology across species. SEQ ID NO: 3 shows the sequence ofthe canine EPO propeptide, with the mature protein beginning at aminoacid 41. The leader sequence is underlined in FIG. 3a . SEQ ID NO: 4shows the sequence of the feline EPO propeptide, with the mature proteinbeginning at amino acid 27. The leader sequence is underlined in FIG.3B.

In one embodiment, functional variants of EPO include variants which mayinclude up to about 10% variation from an EPO nucleic acid or amino acidsequence described herein or known in the art, which retain the functionof the wild type sequence. The sequence on which the EPO variant isbased may, in some embodiments, include the propeptide leader sequence(e.g., as shown in SEQ ID NO: 3 and SEQ ID NO: 4). In anotherembodiment, the EPO variant described herein refers only to the maturepeptide (e.g., amino acids 41-206 of SEQ ID NO: 3 or amino acids 27-192of SEQ ID NO: 4). As used herein, by “retain function” it is meant thatthe nucleic acid or amino acid functions in the same way as the wildtype sequence, although not necessarily at the same level of expressionor activity. For example, in one embodiment, a functional variant hasincreased expression or activity as compared to the wild type sequence.In another embodiment, the functional variant has decreased expressionor activity as compared to the wild type sequence. In one embodiment,the functional variant has 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%or greater increase or decrease in expression or activity as compared tothe wild type sequence. The amino acid sequence of canine EPO isprovided herein as SEQ ID NO: 3. The amino acid sequence of feline EPOis provided herein as SEQ ID NO: 4.

In another embodiment, functional variants of EPO include variants whichmay include up to about 20% variation from an EPO nucleic acid or aminoacid sequence described herein or known in the art, which retain thefunction of the wild type sequence. In one embodiment, functionalvariants of EPO include variants which may include up to about 30%variation from an EPO nucleic acid or amino acid sequence describedherein or known in the art, which retain the function of the wild typesequence. The following alignment shows the canine sequence on top, thefeline sequence on the bottom, with the consensus sequence in themiddle.

Canine (query; amino acids 19 to 206 of SEQ ID NO: 3) v. Feline (subject,  amino acids 5 to 192 of SEQ ID NO: 4) Query 19ECPALLLLLSLLLLPLGLPVLGAPPRLICDSRVLERYILEAREAENVTMGCAQGCSFSEN 78ECPALLLLLSLLLLPLGLPVLGAPPPLICDSRVLERYILEAREAENVTMGCA+GCSFSEN Sbjct 5ECPALLLLLSLLLLPLGLPVLGAPPRLICDSRVLERYILEAREAENVTMGCAEGCSFSEN 64 Query 79ITVPDTKVNFYTWKRMDVGQQALEVWQGLALLSEAILRGQALLANASQPSETPQLHVDKA 136ITVPDTKVNFYTWKRMDVGQQA+EVWQGLALLSEAILRGQALLAN+SQPSET QLHVDKA Sbjct 65ITVPDTKVNFYTWKRMDVGQQAVEVWQGLALLSEAILRGQALLANSSQPSETLQLHVDKA 124 Query139 VSSLRSLTSLLRALGAQKEAMSLPEEASPAPLRTFTVDTLCKLFRIYSNFLRGKLTLYTG 198VSSLRSLTSLLRALGAQKEA SLPE  S APLRTFTVDTLCKLFRIYSNFLRGKLTLYTG Sbjct 125VSSLRSLTSLLRALGAQKEATSLPEATSAAPLRTFTVDTLCKLFRIYSNFLPGKLTLYTG 184 Query199 EACRRGDR 206 EACRRGDR Sbjct 185 EACRRGDR. 192

In one embodiment, the term EPO refers to active EPO in which one ormore amino acid substitutions have been made, as compared to the wildtype sequence (SEQ ID NO: 3 or SEQ ID NO: 4, either sequence with orwithout the leader peptide). In one embodiment, one or more amino acidsubstitutions are made in a residue in which variation is shown acrossspecies as in the alignment above. In another embodiment, one or moreamino acid substitutions are made in a residue in which conservation isshown across species. Although EPO shares a high degree of identityacross species, in one embodiment, it is desirable to select the EPOsequence based on the species of the subject for which administration ofthe vector is ultimately intended. In one example, the subject is amammal. For example, in one embodiment, if the subject is a feline, theEPO sequence is derived from a feline protein. In another embodiment,the EPO sequence is derived from a canine protein. In anotherembodiment, the EPO sequence is derived from a non-human primateprotein. In another embodiment, the EPO is derived from bovine, ovine,or porcine protein. In another embodiment, the EPO sequence is SEQ IDNO: 3. In another embodiment, the EPO sequence is SEQ ID NO: 4.

The EPO peptide or nucleic acid coding sequence may include aheterologous leader sequence in conjunction with the EPO mature proteinsequence. The term “heterologous” when used with reference to a proteinor a nucleic acid indicates that the protein or the nucleic acidcomprises two or more sequences or subsequences which are not found inthe same relationship to each other in nature. For instance, theexpression cassette is typically recombinantly produced, having two ormore sequences from unrelated genes arranged to make a new functionalnucleic acid. For example, in one embodiment, the leader sequence may befrom a different gene than EPO. Thus, with reference to the EPO codingsequences, the leader is heterologous. In one embodiment, the leadersequence is derived from a different species than the EPO sequence.

In one embodiment, the sequence encodes an IL-2 leader peptide fusedupstream of the EPO mature polypeptide. In one embodiment, the leadersequence is SEQ ID NO: 9: M Y R M Q L L S C I A L S L A L V T N S.However, another heterologous leader sequence may be substituted for theIL-2 signal/leader peptide. The leader may be a signal sequence which isnatively found in a cytokine (e.g., IL-2, IL12, IL18, or the like),immunoglobulin, insulin, albumin, β-glucuronidase, alkaline protease orthe fibronectin secretory signal peptides, or sequences from tissuespecific secreted proteins, amongst others. In one embodiment, theleader sequence is the endogenous leader sequence from the EPOpropeptide.

As used herein, the terms “derived” or “derived from” mean the sequenceor protein is sourced from a specific subject species or shares the samesequence as a protein or sequence sourced from a specific subjectspecies. For example, a propeptide sequence which is “derived from” acanine, shares the same sequence (or a variant thereof, as definedherein) as the same propeptide sequence as expressed in a canine.However, the specified nucleic acid or amino acid need not actually besourced from a canine. Various techniques are known in the art which areable to produce a desired sequence, including mutagenesis of a similarprotein (e.g., a homolog) or artificial production of a nucleic acid oramino acid sequence. The “derived” nucleic acid or amino acid retainsthe function of the same nucleic acid or amino acid in the species fromwhich it is “derived”, regardless of actual source of the derivedsequence.

As used herein the terms “EPO construct”, “EPO expression construct” andsynonyms include the EPO sequence as described herein. The terms “EPOconstruct”, “EPO expression construct” and synonyms can be used to referto the nucleic acid sequences encoding the EPO (including the EPO matureprotein or propeptide with endogenous or heterologous leader) or theexpression products thereof.

The term “amino acid substitution” and its synonyms are intended toencompass modification of an amino acid sequence by replacement of anamino acid with another, substituting, amino acid. The substitution maybe a conservative substitution. It may also be a non-conservativesubstitution. The term conservative, in referring to two amino acids, isintended to mean that the amino acids share a common property recognizedby one of skill in the art. For example, amino acids having hydrophobicnonacidic side chains, amino acids having hydrophobic acidic sidechains, amino acids having hydrophilic nonacidic side chains, aminoacids having hydrophilic acidic side chains, and amino acids havinghydrophilic basic side chains. Common properties may also be amino acidshaving hydrophobic side chains, amino acids having aliphatic hydrophobicside chains, amino acids having aromatic hydrophobic side chains, aminoacids with polar neutral side chains, amino acids with electricallycharged side chains, amino acids with electrically charged acidic sidechains, and amino acids with electrically charged basic side chains.Both naturally occurring and non-naturally occurring amino acids areknown in the art and may be used as substituting amino acids inembodiments. Methods for replacing an amino acid are well known to theskilled in the art and include, but are not limited to, mutations of thenucleotide sequence encoding the amino acid sequence. Reference to “oneor more” herein is intended to encompass the individual embodiments of,for example, 1, 2, 3, 4, 5, 6, or more.

Also provided are the assembled EPO proteins described herein. In oneembodiment, the EPO protein is produced by a described AAV construct. Inone embodiment, the EPO protein includes a heterologous leader combinedwith the mature EPO protein. In one embodiment, the heterologous leaderis from IL-2. The assembled EPO proteins have many uses includingdiagnostic assays. Thus, in one embodiment, the EPO protein is labeled.As used herein, “labels” are chemical or biochemical moieties useful forlabeling the EPO protein. “Labels” include fluorescent agents,chemiluminescent agents, chromogenic agents, quenching agents,radionucleotides, enzymes, substrates, cofactors, inhibitors,radioactive isotopes, magnetic particles, and other moieties known inthe art. “Labels” or “reporter molecules” are capable of generating ameasurable signal and may be covalently or noncovalently joined to anoligonucleotide or nucleotide (e.g., a non-natural nucleotide) orligand. Most desirably, the label is detectable visually, e.g.colorimetrically. Many such labels are known in the art and include,without limitation, fluorescent detectable fluorochromes, e.g.,fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin(APC), coriphosphine-O (CPO) or tandem dyes, PE-cyanin-5 (PC5), andPE-Texas Red (ECD). Commonly used fluorochromes include fluoresceinisothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), andalso include the tandem dyes, PE-cyanin-5 (PC5), PE-cyanin-7 (PC7),PE-cyanin-5.5, PE-Texas Red (ECD), rhodamine, PerCP, fluoresceinisothiocyanate (FITC) and Alexa dyes. Combinations of such labels, suchas Texas Red and rhodamine, FITC+PE, FITC+PECy5 and PE+PECy7, amongothers may be used depending upon assay method. Other desirable labelsor tags include those which allow physical separation or immobilizationon a substrate of the protein. Such labels include biotin. Othersuitable labels or tags are described, e.g., in US 2011-0177967 A1,which is incorporated herein by reference.

In another embodiment, the EPO peptide includes variants which mayinclude up to about 10% variation from the EPO sequence. That is, theEPO peptide shares about 90% identity to about 99.9% identity, about 95%to about 99% identity or about 97% to about 98% identity to the EPOsequences provided herein and/or known in the art.

In addition to the EPO peptides provided herein, nucleic acid sequencesencoding these peptides are provided. In one embodiment, a nucleic acidsequence is provided which encodes for the EPO peptides describedherein. In another embodiment, this includes any nucleic acid sequencewhich encodes the canine EPO protein of SEQ ID NO: 3 or a sequencesharing at least 90% identity with SEQ ID NO: 3. In another embodiment,this includes any nucleic acid sequence which encodes the feline EPOprotein of SEQ ID NO: 4 or a sequence sharing at least 90% identity withSEQ ID NO: 4.

In one embodiment, the nucleic acid sequence encoding canine EPO is SEQID NO: 5. In one embodiment, the nucleic acid sequence encoding felineEPO is SEQ ID NO: 6. In yet another embodiment, the EPO nucleic acidincludes variants which may include up to about 10% variation from anEPO sequence disclosed herein or known in the art. In yet anotherembodiment, the EPO nucleic acid includes variants which may include upto about 20% variation from an EPO sequence disclosed herein or known inthe art. In yet another embodiment, the EPO nucleic acid includesvariants which may include up to about 30% variation from an EPOsequence disclosed herein or known in the art. In another embodiment,the EPO nucleic acid includes variants which may include up to about 40%variation from an EPO sequence disclosed herein or known in the art.

In one embodiment, the nucleic acid sequence encoding EPO is a codonoptimized sequence encoding any of the EPO peptides described herein,including sequences sharing at least 90% identity with the describedsequence. In one embodiment, the nucleic acid sequence is codonoptimized for expression in the subject for which administration isdesired. In one embodiment, the nucleic acid sequence encoding canineEPO is SEQ ID NO: 7. In one embodiment, the nucleic acid sequenceencoding feline EPO is SEQ ID NO: 8.

When a variant of the EPO peptide is desired, the coding sequences forthese peptides may be generated using site-directed mutagenesis of thewild-type nucleic acid sequence. Web-based or commercially availablecomputer programs, as well as service based companies may be used toback translate the amino acids sequences to nucleic acid codingsequences, including both RNA and/or cDNA. See, e.g., backtranseq byEMBOSS, http://www.ebi.ac.uk/Tools/st/; Gene Infinity(http://www.geneinfinity.org/sms-/sms_backtranslation.html); ExPasy(http://www.expasy.org/tools/). In one embodiment, the RNA and/or cDNAcoding sequences are designed for optimal expression in the subjectspecies for which administration is ultimately intended, as discussedherein. Thus, in one embodiment, the coding sequences are designed foroptimal expression in a feline. In another embodiment, the codingsequences are designed for optimal expression in a canine. In yetanother embodiment, the coding sequences are designed for optimalexpression in a primate.

The coding sequences may be designed for optimal expression using codonoptimization. Codon-optimized coding regions can be designed by variousdifferent methods. This optimization may be performed using methodswhich are available on-line, published methods, or a company whichprovides codon optimizing services. One codon optimizing method isdescribed, e.g., in International Patent Publication No. WO 2015/012924,which is incorporated by reference herein. Briefly, the nucleic acidsequence encoding the product is modified with synonymous codonsequences. Suitably, the entire length of the open reading frame (ORF)for the product is modified. However, in some embodiments, only afragment of the ORF may be altered. By using one of these methods, onecan apply the frequencies to any given polypeptide sequence, and producea nucleic acid fragment of a codon-optimized coding region which encodesthe polypeptide.

The terms “percent (%) identity”, “sequence identity”, “percent sequenceidentity”, or “percent identical” in the context of nucleic acidsequences refers to the bases in the two sequences which are the samewhen aligned for correspondence. The length of sequence identitycomparison may be over the full-length of the genome, the full-length ofa gene coding sequence, or a fragment of at least about 100 to 150nucleotides, or as desired. However, identity among smaller fragments,e.g. of at least about nine nucleotides, usually at least about 20 to 24nucleotides, at least about 28 to 32 nucleotides, at least about 36 ormore nucleotides, may also be desired. Multiple sequence alignmentprograms are also available for nucleic acid sequences. Examples of suchprograms include, “Clustal W”, “CAP Sequence Assembly”, “BLAST”, “MAP”,and “MEME”, which are accessible through Web Servers on the internet.Other sources for such programs are known to those of skill in the art.Alternatively, Vector NTI utilities are also used. There are also anumber of algorithms known in the art that can be used to measurenucleotide sequence identity, including those contained in the programsdescribed above. As another example, polynucleotide sequences can becompared using Fasta™, a program in GCG Version 6.1. Fasta™ providesalignments and percent sequence identity of the regions of the bestoverlap between the query and search sequences. For instance, percentsequence identity between nucleic acid sequences can be determined usingFasta™ with its default parameters (a word size of 6 and the NOPAMfactor for the scoring matrix) as provided in GCG Version 6.1, hereinincorporated by reference.

The terms “percent (%) identity”, “sequence identity”, “percent sequenceidentity”, or “percent identical” in the context of amino acid sequencesrefers to the residues in the two sequences which are the same whenaligned for correspondence. Percent identity may be readily determinedfor amino acid sequences over the full-length of a protein, polypeptide,about 70 amino acids to about 100 amino acids, or a peptide fragmentthereof or the corresponding nucleic acid sequence coding sequencers. Asuitable amino acid fragment may be at least about 8 amino acids inlength, and may be up to about 150 amino acids. Generally, whenreferring to “identity”, “homology”, or “similarity” between twodifferent sequences, “identity”, “homology” or “similarity” isdetermined in reference to “aligned” sequences. “Aligned” sequences or“alignments” refer to multiple nucleic acid sequences or protein (aminoacids) sequences, often containing corrections for missing or additionalbases or amino acids as compared to a reference sequence. Alignments areperformed using any of a variety of publicly or commercially availableMultiple Sequence Alignment Programs. Sequence alignment programs areavailable for amino acid sequences, e.g., the “Clustal X”, “MAP”,“PIMA”, “MSA”, “BLOCKMAKER”, “MEME”, and “Match-Box” programs.Generally, any of these programs are used at default settings, althoughone of skill in the art can alter these settings as needed.Alternatively, one of skill in the art can utilize another algorithm orcomputer program which provides at least the level of identity oralignment as that provided by the referenced algorithms and programs.See, e.g., J. D. Thomson et al, Nucl. Acids. Res., “A comprehensivecomparison of multiple sequence alignments”, 27(13):2682-2690 (1999).

In one embodiment, the nucleic acid sequences encoding the EPOconstructs described herein are engineered into any suitable geneticelement, e.g., naked DNA, phage, transposon, cosmid, RNA molecule (e.g.,mRNA), episome, etc., which transfers the EPO sequences carried thereonto a host cell, e.g., for generating nanoparticles carrying DNA or RNA,viral vectors in a packaging host cell and/or for delivery to a hostcells in subject. In one embodiment, the genetic element is a plasmid.The selected genetic element may be delivered by any suitable method,including transfection, electroporation, liposome delivery, membranefusion techniques, high velocity DNA-coated pellets, viral infection andprotoplast fusion. The methods used to make such constructs are known tothose with skill in nucleic acid manipulation and include geneticengineering, recombinant engineering, and synthetic techniques. See,e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Press, Cold Spring Harbor, N.Y. (2012).

As used herein, an “expression cassette” refers to a nucleic acidmolecule which comprises coding sequences for the EPO peptide, promoter,and may include other regulatory sequences therefor, which cassette maybe engineered into a genetic element and/or packaged into the capsid ofa viral vector (e.g., a viral particle). Typically, such an expressioncassette for generating a viral vector contains the EPO constructsequences described herein flanked by packaging signals of the viralgenome and other expression control sequences such as those describedherein. Any of the expression control sequences can be optimized for aspecific species using techniques known in the art including, e.g.,codon optimization, as described herein.

The expression cassette typically contains a promoter sequence as partof the expression control sequences. In one embodiment, a CB7 promoteris used. CB7 is a chicken β-actin promoter with cytomegalovirus enhancerelements. Alternatively, other liver-specific promoters may be used[see, e.g., The Liver Specific Gene Promoter Database, Cold SpringHarbor, available online at rulai.schl.edu/LSPD, alpha 1 anti-trypsin(A1AT); human albumin Miyatake et al., J. Virol., 71:5124 32 (1997),humAlb; and hepatitis B virus core promoter, Sandig et al., Gene Ther.,3:1002 9 (1996)]. TTR minimal enhancer/promoter, alpha-antitrypsinpromoter, LSP (845 nt)25 (requires intron-less scAAV). In oneembodiment, the liver-specific promoter thyroxin binding globulin (TBG)is used. Other promoters, such as viral promoters, constitutivepromoters, regulatable promoters [see, e.g., WO 2011/126808 and WO2013/04943], or a promoter responsive to physiologic cues may be usedmay be utilized in the vectors described herein.

In addition to a promoter, an expression cassette and/or a vector maycontain other appropriate transcription initiation, termination,enhancer sequences, efficient RNA processing signals such as splicingand polyadenylation (polyA) signals; TATA sequences; sequences thatstabilize cytoplasmic mRNA; sequences that enhance translationefficiency (i.e., Kozak consensus sequence); introns; sequences thatenhance protein stability; and when desired, sequences that enhancesecretion of the encoded product. The expression cassette or vector maycontain none, one or more of any of the elements described herein.Examples of suitable polyA sequences include, e.g., SV40, bovine growthhormone (bGH), and TK polyA. Examples of suitable enhancers include,e.g., the CMV enhancer, the RSV enhancer, the alpha fetoproteinenhancer, the TTR minimal promoter/enhancer, LSP (TH-binding globulinpromoter/alpha1-microglobulin/bikunin enhancer), amongst others.

In one embodiment, the viral vector includes a nucleic acid expressioncassette comprising: a 5′ AAV inverted terminal repeat sequence (ITR), apromoter with optional enhancer, an EPO sequence, a poly A sequence, anda 3′ AAV ITR, wherein said expression cassette expresses a functionalEPO in a host cell.

These control sequences are “operably linked” to the EPO constructsequences. As used herein, the term “operably linked” refers to bothexpression control sequences that are contiguous with the gene ofinterest and expression control sequences that act in trans or at adistance to control the gene of interest.

The expression cassette may be engineered onto a plasmid which is usedfor production of a viral vector. The minimal sequences required topackage the expression cassette into an AAV viral particle are the AAV5′ and 3′ ITRs, which may be of the same AAV origin as the capsid, or ofa different AAV origin (to produce an AAV pseudotype). In oneembodiment, the ITR sequences from AAV2, or the deleted version thereof(ΔITR), are used for convenience and to accelerate regulatory approval.However, ITRs from other AAV sources may be selected. Where the sourceof the ITRs is from AAV2 and the AAV capsid is from another AAV source,the resulting vector may be termed pseudotyped. Typically, an expressioncassette for an AAV vector comprises an AAV 5′ ITR, the propeptide-EPOactive peptide coding sequences and any regulatory sequences, and an AAV3′ ITR. However, other configurations of these elements may be suitable.A shortened version of the 5′ ITR, termed ΔITR, has been described inwhich the D-sequence and terminal resolution site (trs) are deleted. Inother embodiments, the full-length AAV 5′ and 3′ ITRs are used.

Exemplary plasmids are provided in the sequence listing. SEQ ID NO: 1provides the sequence of a plasmid encoding a canine EPO construct,entitled pn1044.CB7.caEPO. In one embodiment, the expression cassette isengineered into the plasmid of SEQ ID NO: 1. SEQ ID NO: 2 provides thesequence of a plasmid encoding a feline EPO construct, entitledpn1044.CB7.feEPO. In one embodiment, the expression cassette isengineered into the plasmid of SEQ ID NO: 2. Plasmids, such as those,shown in SEQ ID NO: 1 and SEQ ID NO: 2 may be modified to include one ormore additional components as described herein, or to remove or replacecomponents as necessary. In one embodiment, the plasmid has the sequenceof SEQ ID NO: 1 or a sequence sharing at least 80% identity therewith.In another embodiment, the plasmid has the sequence of SEQ ID NO: 2 or asequence sharing at least 80% identity therewith.

The abbreviation “sc” refers to self-complementary. “Self-complementaryAAV” refers a plasmid or vector having an expression cassette in which acoding region carried by a recombinant AAV nucleic acid sequence hasbeen designed to form an intra-molecular double-stranded DNA template.Upon infection, rather than waiting for cell mediated synthesis of thesecond strand, the two complementary halves of scAAV will associate toform one double stranded DNA (dsDNA) unit that is ready for immediatereplication and transcription. See, e.g., D M McCarty et al,“Self-complementary recombinant adeno-associated virus (scAAV) vectorspromote efficient transduction independently of DNA synthesis”, GeneTherapy, (August 2001), Vol 8, Number 16, Pages 1248-1254.Self-complementary AAVs are described in, e.g., U.S. Pat. Nos.6,596,535; 7,125,717; and 7,456,683, each of which is incorporatedherein by reference in its entirety.

An adeno-associated virus (AAV) viral vector is an AAV DNase-resistantparticle having an AAV protein capsid into which is packaged nucleicacid sequences for delivery to target cells. An AAV capsid is composedof 60 capsid (cap) protein subunits, VP1, VP2, and VP3, that arearranged in an icosahedral symmetry in a ratio of approximately 1:1:10to 1:1:20, depending upon the selected AAV. AAV serotypes may beselected as sources for capsids of AAV viral vectors (DNase resistantviral particles) including, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6,AAV6.2, AAV7, AAV8, AAV9, rh10, AAVrh64R1, AAVrh64R2, rh8, rh.10,variants of any of the known or mentioned AAVs or AAVs yet to bediscovered. In one embodiment, the AAV is an AAV8 capsid, or a variantthereof. See, e.g., US Published Patent Application No. 2007-0036760-A1;US Published Patent Application No. 2009-0197338-A1; EP 1310571. Seealso, WO 2003/042397 (AAV7 and other simian AAV), U.S. Pat. Nos.7,790,449 and 7,282,199 (AAV8), WO 2005/033321 and U.S. Pat. No.7,906,111 (AAV9), and WO 2006/110689, and WO 2003/042397 (rh.10).Alternatively, a recombinant AAV based upon any of the recited AAVs, maybe used as a source for the AAV capsid. These documents also describeother AAV which may be selected for generating AAV and are incorporatedby reference. In some embodiments, an AAV cap for use in the viralvector can be generated by mutagenesis (i.e., by insertions, deletions,or substitutions) of one of the aforementioned AAV Caps or its encodingnucleic acid. In some embodiments, the AAV capsid is chimeric,comprising domains from two or three or four or more of theaforementioned AAV capsid proteins. In some embodiments, the AAV capsidis a mosaic of Vp1, Vp2, and Vp3 monomers from two or three differentAAVs or recombinant AAVs. In some embodiments, an rAAV compositioncomprises more than one of the aforementioned Caps. In anotherembodiment, the AAV capsid includes variants which may include up toabout 10% variation from any described or known AAV capsid sequence.That is, the AAV capsid shares about 90% identity to about 99.9%identity, about 95% to about 99% identity or about 97% to about 98%identity to an AAV capsid provided herein and/or known in the art. Inone embodiment, the AAV capsid shares at least 95% identity with an AAVcapsid. When determining the percent identity of an AAV capsid, thecomparison may be made over any of the variable proteins (e.g., vp1,vp2, or vp3). In one embodiment, the AAV capsid shares at least 95%identity with the AAV8 vp3. In another embodiment, a self-complementaryAAV is used.

For packaging an expression cassette into virions, the ITRs are the onlyAAV components required in cis in the same construct as the gene. In oneembodiment, the coding sequences for the replication (rep) and/or capsid(cap) are removed from the AAV genome and supplied in trans or by apackaging cell line in order to generate the AAV vector. For example, asdescribed above, a pseudotyped AAV may contain ITRs from a source whichdiffers from the source of the AAV capsid. Additionally oralternatively, a chimeric AAV capsid may be utilized. Still other AAVcomponents may be selected. Sources of such AAV sequences are describedherein and may also be isolated or obtained from academic, commercial,or public sources (e.g., the American Type Culture Collection, Manassas,Va.). Alternatively, the AAV sequences may be obtained through syntheticor other suitable means by reference to published sequences such as areavailable in the literature or in databases such as, e.g., GenBank®,PubMed®, or the like.

Methods for generating and isolating AAV viral vectors suitable fordelivery to a subject are known in the art. See, e.g., U.S. Pat. Nos.7,790,449; 7,282,199; WO 2003/042397; WO 2005/033321, WO 2006/110689;and U.S. Pat. No. 7,588,772 B2]. In a one system, a producer cell lineis transiently transfected with a construct that encodes the transgeneflanked by ITRs and a construct(s) that encodes rep and cap. In a secondsystem, a packaging cell line that stably supplies rep and cap istransiently transfected with a construct encoding the transgene flankedby ITRs. In each of these systems, AAV virions are produced in responseto infection with helper adenovirus or herpesvirus, requiring theseparation of the rAAVs from contaminating virus. More recently, systemshave been developed that do not require infection with helper virus torecover the AAV—the required helper functions (i.e., adenovirus E1, E2a,VA, and E4 or herpesvirus UL5, UL8, UL52, and UL29, and herpesviruspolymerase) are also supplied, in trans, by the system. In these newersystems, the helper functions can be supplied by transient transfectionof the cells with constructs that encode the required helper functions,or the cells can be engineered to stably contain genes encoding thehelper functions, the expression of which can be controlled at thetranscriptional or posttranscriptional level. In yet another system, thetransgene flanked by ITRs and rep/cap genes are introduced into insectcells by infection with baculovirus-based vectors. For reviews on theseproduction systems, see generally, e.g., Zhang et al., 2009,“Adenovirus-adeno-associated virus hybrid for large-scale recombinantadeno-associated virus production,” Human Gene Therapy 20:922-929, thecontents of each of which is incorporated herein by reference in itsentirety. Methods of making and using these and other AAV productionsystems are also described in the following U.S. patents, the contentsof each of which is incorporated herein by reference in its entirety:U.S. Pat. Nos. 5,139,941; 5,741,683; 6,057,152; 6,204,059; 6,268,213;6,491,907; 6,660,514; 6,951,753; 7,094,604; 7,172,893; 7,201,898;7,229,823; and 7,439,065. See generally, e.g., Grieger & Samulski, 2005,“Adeno-associated virus as a gene therapy vector: Vector development,production and clinical applications,” Adv. Biochem. Engin/Biotechnol.99: 119-145; Buning et al., 2008, “Recent developments inadeno-associated virus vector technology,” J. Gene Med. 10:717-733; andthe references cited below, each of which is incorporated herein byreference in its entirety. The methods used to construct any embodimentof this invention are known to those with skill in nucleic acidmanipulation and include genetic engineering, recombinant engineering,and synthetic techniques. See, e.g., Green and Sambrook et al, MolecularCloning: A Laboratory Manual, Cold Spring Harbor Press, Cold SpringHarbor, N.Y. (2012). Similarly, methods of generating rAAV virions arewell known and the selection of a suitable method is not a limitation onthe present invention. See, e.g., K. Fisher et al, (1993) J. Virol.,70:520-532 and U.S. Pat. No. 5,478,745.

Also provided are compositions which include the viral vector constructsdescribed herein. The pharmaceutical compositions described herein aredesigned for delivery to subjects in need thereof by any suitable routeor a combination of different routes. Direct delivery to the liver(optionally via intravenous, via the hepatic artery, or by transplant),oral, inhalation, intranasal, intratracheal, intraarterial, intraocular,intravenous, intramuscular, subcutaneous, intradermal, and otherparental routes of administration. The viral vectors described hereinmay be delivered in a single composition or multiple compositions.Optionally, two or more different AAV may be delivered, or multipleviruses [see, e.g., WO 2011/126808 and WO 2013/049493]. In anotherembodiment, multiple viruses may contain different replication-defectiveviruses (e.g., AAV and adenovirus).

The replication-defective viruses can be formulated with aphysiologically acceptable carrier for use in gene transfer and genetherapy applications. In the case of AAV viral vectors, quantificationof the genome copies (“GC”) may be used as the measure of the dosecontained in the formulation or suspension. Any method known in the artcan be used to determine the genome copy (GC) number of thereplication-defective virus compositions of the invention. One methodfor performing AAV GC number titration is as follows: Purified AAVvector samples are first treated with DNase to eliminate un-encapsidatedAAV genome DNA or contaminating plasmid DNA from the production process.The DNase resistant particles are then subjected to heat treatment torelease the genome from the capsid. The released genomes are thenquantitated by real-time PCR using primer/probe sets targeting specificregion of the viral genome (usually poly A signal).

Also, the replication-defective virus compositions can be formulated indosage units to contain an amount of replication-defective virus that isin the range of about 1.0×10⁹ GC to about 1.0×10¹⁵ GC. In anotherembodiment, this amount of viral genome may be delivered in split doses.In one embodiment, the dosage is about 1.0×10¹⁰ GC to about 1.0×10¹² GCfor an average feline or small canine subject of about 5 kg. In oneembodiment, the dosage is about 1.0×10¹¹ GC to about 1.0×10¹³ GC for anaverage medium canine subject of about 20 kg. The average canine rangesfrom about 5 to about 50 kg in body weight. In one embodiment, thedosage is about 1.0×10¹¹ GC to 1.0×10¹³ GC for a subject. In anotherembodiment, the dose about 3×10¹² GC. For example, the dose of AAV virusmay be about 1×10¹¹ GC, about 5×10¹¹ GC, about 1×10¹² GC, about 5×10¹²GC, or about 1×10¹³ GC. In one embodiment, the dosage is about 3×10¹⁰GC/kg. In another example, the constructs may be delivered in an amountof about 0.001 mg to about 10 mg per mL. In one embodiment, theconstructs may be delivered in volumes from 1 μL to about 100 mL for aveterinary subject. See, e.g., Diehl et al, J. Applied Toxicology,21:15-23 (2001) for a discussion of good practices for administration ofsubstances to various veterinary animals. This document is incorporatedherein by reference. As used herein, the term “dosage” can refer to thetotal dosage delivered to the subject in the course of treatment, or theamount delivered in a single (of multiple) administration.

The above-described recombinant vectors may be delivered to host cellsaccording to published methods. The rAAV, preferably suspended in aphysiologically compatible carrier, diluent, excipient and/or adjuvant,may be administered to a desired subject including without limitation, acat, dog, or other non-human mammalian subject. Suitable carriers may bereadily selected by one of skill in the art in view of the indicationfor which the transfer virus is directed. For example, one suitablecarrier includes saline, which may be formulated with a variety ofbuffering solutions (e.g., phosphate buffered saline). Other exemplarycarriers include sterile saline, lactose, sucrose, calcium phosphate,gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water. Theselection of the carrier is not a limitation of the present invention.

Optionally, the compositions of the invention may contain, in additionto the rAAV and/or variants and carrier(s), other conventionalpharmaceutical ingredients, such as preservatives, or chemicalstabilizers. Suitable exemplary preservatives include chlorobutanol,potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, theparabens, ethyl vanillin, glycerin, phenol, and parachlorophenol.Suitable chemical stabilizers include gelatin and albumin.

The viral vectors and other constructs described herein may be used inpreparing a medicament for delivering an EPO construct to a subject inneed thereof and/or for treating chronic kidney disease in a subject.Thus, in another aspect a method of treating chronic kidney disease isprovided. The method includes administering a composition as describedherein to a subject in need thereof. In one embodiment, the compositionincludes a viral vector containing an EPO expression cassette, asdescribed herein. In one embodiment, the subject is a mammal. In anotherembodiment, the subject is a feline or canine. In yet another aspect amethod of treating anemia is provided. The method includes administeringa composition as described herein to a subject in need thereof. In oneembodiment, the composition includes a viral vector containing an EPOexpression cassette, as described herein. In one embodiment, the subjectis a mammal. In another embodiment, the subject is a feline or canine.

In another embodiment, a method for treating chronic kidney disease in afeline is provided. The method includes administering an AAV viralvector comprising a nucleic acid molecule comprising a sequence encodingfeline EPO. In another embodiment, a method for treating chronic kidneydisease in a canine is provided. The method includes administering anAAV viral vector comprising a nucleic acid molecule comprising asequence encoding canine EPO.

A course of treatment may optionally involve repeat administration ofthe same viral vector (e.g., an AAV8 vector) or a different viral vector(e.g., an AAV8 and an AAVrh10). Still other combinations may be selectedusing the viral vectors described herein. Optionally, the compositiondescribed herein may be combined in a regimen involving other drugs orprotein-based therapies, including e.g., recombinant EPO. Optionally,the composition described herein may be combined in a regimen involvinglifestyle changes including dietary and exercise regimens.

It is to be noted that the term “a” or “an” refers to one or more. Assuch, the terms “a” (or “an”), “one or more,” and “at least one” areused interchangeably herein.

The words “comprise”, “comprises”, and “comprising” are to beinterpreted inclusively rather than exclusively. The words “consist”,“consisting”, and its variants, are to be interpreted exclusively,rather than inclusively. While various embodiments in the specificationare presented using “comprising” language, under other circumstances, arelated embodiment is also intended to be interpreted and describedusing “consisting of” or “consisting essentially of” language.

As used herein, the term “about” means a variability of 10% from thereference given, unless otherwise specified.

The term “regulation” or variations thereof as used herein refers to theability of a composition to inhibit one or more components of abiological pathway.

A “subject” is a mammal, e.g., a human, mouse, rat, guinea pig, dog,cat, horse, cow, pig, or non-human primate, such as a monkey,chimpanzee, baboon or gorilla. As used herein, the term “subject” isused interchangeably with “patient”.

As used herein, “disease”, “disorder” and “condition” are usedinterchangeably, to indicate an abnormal state in a subject.

Unless defined otherwise in this specification, technical and scientificterms used herein have the same meaning as commonly understood by one ofordinary skill in the art and by reference to published texts, whichprovide one skilled in the art with a general guide to many of the termsused in the present application.

The following examples are illustrative only and are not intended tolimit the present invention.

Example 1 Construction of EPO Vectors

The amino acid sequences of canine and feline erythropoietin wereobtained from Genbank. The amino acid sequences were backtranslated andcodon optimized, followed by addition of a kozac consensus sequence,stop codon, and cloning sites. The sequences were produced by GeneArt,and cloned into an expression vector containing a chicken-beta actinpromoter with CMV enhancer (p1044). The expression construct is flankedby AAV2 ITRs. The canine and feline constructs were packaged in an AAVserotype 8 capsid by triple transfection and iodixanol gradientpurification and titered by Taqman quantitative PCR.

Example 2 AAV-Mediated Expression of Feline Erythropoietin in Cats

Three cats were treated with a single intramuscular injection of 3×10¹⁰genome copies per kilogram body weight (GC/kg) AAV8 expressing felineerythropoietin (FIG. 1). Blood samples were collected at the time ofinjection and periodically thereafter for measurement of hematocrit.Therapeutic phlebotomy was initiated on day 42 after vector injection.To date, the results have shown seen sustained expression of EPO forgreater than 100 days.

Example 3 AAV-Mediated Expression of Canine Erythropoietin in Dogs

Three dogs were treated with a single intramuscular injection of 3×10¹⁰GC/kg AAV8 expressing canine erythropoietin. Blood samples werecollected at the time of injection and periodically thereafter formeasurement of hematocrit (FIG. 2). Blood samples from untreatedlittermates were included as controls. Therapeutic phlebotomy wasinitiated on day 60 after vector injection.

Example 4 Dosage Study of AAV-Mediated Expression of FelineErythropoietin in Cats

Cats were treated with a single intramuscular injection of up to 3×10⁷,3×10⁸, 3×10⁹, or 3×10¹⁰ genome copies per kilogram body weight (GC/kg)of AAV8 expressing feline erythropoietin. These three cohorts were of 4cats per dosage. All cats were normal/wildtype and selected randomly.The purpose of this study was show long term safety and efficacy tohighlight a possible clinical candidate for a client owned animal study.FIG. 4.

Example 5 AAV-Mediated Expression of Feline Erythropoietin in Cats

Cats are treated with a single intramuscular injection of up to 3×10⁹genome copies per kilogram body weight (GC/kg) AAV8 expressing felineerythropoietin in the left or right quadriceps in a total volume of upto 400 μL. Blood samples are collected at the time of injection andperiodically thereafter for measurement of hematocrit. Vector may bereadministered 28 days or more post the initial vector administrationusing the same criteria.

This study will include up to nine cats with anemia related to stage IIIchronic kidney disease. CKD related anemia will be defined as ahematocrit less than 29% on two occasions at least one month apart andwithout another apparent cause for the anemia. Enrolled subjects willreceive a single intramuscular injection of an adenoassociated virusvector carrying a feline erythropoietin transgene (AAV8.fEpo). Subjectswill be evaluated at the study site at the time of vector administrationand at 2, 4, 6, and 8 weeks after administration. At each visit bloodwill be collected for evaluation of erythropoietin concentration,hematocrit, reticulocyte count, mean corpuscular volume, meancorpuscular hemoglobin, and mean corpuscular hemoglobin concentration.Patients will return to the study site or follow up with their primaryveterinarian at 3 months, 6 months and 12 months after study drugadministration for measurement of hematocrit. The study drug haspreviously been found to be safe in 4 normal cats at doses up to 3E8genome copies/kg. The first three subjects enrolled in this trial willreceive a dose of 1E8 genome copies/kg AAV8.fEpo. An initial evaluationof safety and vector activity will take place after the first threesubjects have reached 8 weeks post vector administration. Depending onthe results at the 8 week analysis in this initial cohort of 3 animals,dosing of the second cohort of three animals will proceed using thefollowing scheme:

-   -   1. If any severe adverse events occur in the initial cohort or        if any animal reaches a hematocrit 55%, the dose will be reduced        threefold and three additional animals will be enrolled at this        reduced dose. (total study enrollment of 6 subjects)    -   2. If there are no adverse events and all cats demonstrate an        increase in hematocrit of at least 5% or reach the normal        hematocrit range, 3 additional cats will be treated at the        starting dose. (total study enrollment of 6 subjects)    -   3. If there are no adverse events and all cats do not achieve at        least a 5% increase in hematocrit or reach the normal range, 3        additional cats will be treated at a 3fold higher dose of 3E8        genome copies/kg. There will again be an interim evaluation of        safety and activity after all three animals in this cohort have        reached 6 weeks post vector administration. If there are no        adverse events and all cats demonstrate an increase in        hematocrit of at least 5% or reach the normal hematocrit range,        3 additional cats will be treated at this dose. If there are no        adverse events and all cats do not achieve at least a 5%        increase in hematocrit or reach the normal range, 3 additional        cats will be treated at a dose of 6E8 genome copies/kg. (total        study enrollment of 9 subjects) This study will therefore enroll        at least 6 and as many as 9 subjects depending on the results of        interim analyses of safety and change in hematocrit. Primary        endpoints include safety and evaluation of vector expression    -   Secondary endpoints include quality of life for animals and        long-term sustained expression of vector    -   Inclusion criteria:    -   Cats with stage III renal failure (serum creatinine 2.95 mg/dL)    -   Hematocrit 29% on two occasions at least one month apart    -   Owner willing to return to study site for visits at 2 weeks, 4        weeks, 6 weeks, and 8 weeks after study drug administration, and        to study site or primary veterinary clinic at 3 months, 6 months        and 12 months after study drug administration.    -   Exclusion criteria:    -   Anticipated life expectancy less than 3 months    -   Kidney transplant    -   Past treatment with recombinant erythropoietin (epoetin,        darbepoietin)    -   Any other condition that in the opinion of the principle        investigator would preclude evaluation of the safety and        activity of the study drug    -   Preexisting neutralizing antibodies to AAV8    -   Eligible cats will be screened during first visit. This will        include a full history and clinical exam, CBC/chemistry, and        preexisting antibodies to AAV8, and full release on consent        form.    -   All eligible cats that agree to terms of research protocol will        receive a single intramuscular injection of AAV8.fEPO of up to        3E9 genome copies/kg at least one week after being accepted into        study.    -   Following vector administration, cats will be evaluated every        other week for a duration of 8 weeks. These clinical check ups        will include CBC retic/chem, full clinical evaluation, and serum        collection. After 8 weeks, these clinical evaluations will move        to every 3 months, starting on day 90 after vector        administration. The 3 month, 6 month, 9 month, and 1 year        evaluations will be done.    -   Possible Complications    -   Any animal that displays polycythemia of 65% Hematocrit will be        given a therapeutic phlebotomy of up to 10% blood volume per        every 3 weeks.

All publications cited in this specification, as well as provisionalpatent application Nos. 62/212,144 and 62/336,211 are incorporatedherein by reference. Similarly, the SEQ ID NOs which are referencedherein and which appear in the appended Sequence Listing areincorporated by reference. While the invention has been described withreference to particular embodiments, it will be appreciated thatmodifications can be made without departing from the spirit of theinvention. Such modifications are intended to fall within the scope ofthe appended claims.

What is claimed is:
 1. A recombinant adeno-associated virus (rAAV)comprising an AAV capsid having packaged therein a vector genome,wherein said vector genome comprises a nucleotide sequence encoding afunctional feline erythropoietin (EPO), inverted terminal repeatsequences, and expression control sequences that direct expression ofthe EPO in a host cell, wherein the nucleotide sequence encoding thefeline EPO comprises nucleotides 79 to 576 of the nucleotide sequence ofSEQ ID NO: 8 and encodes at least amino acids 27 to 192 of the aminoacid sequence of SEQ ID NO:
 4. 2. The rAAV of claim 1, wherein theencoded feline EPO comprises the amino acids 27 to 192 of SEQ ID NO: 4in combination with a heterologous leader sequence.
 3. The rAAV of claim1, wherein the EPO nucleotide sequence encodes amino acids 1 to 192 ofSEQ ID NO:
 4. 4. The rAAV of claim 3, wherein the nucleotide sequenceencoding the feline EPO comprises SEQ ID NO:
 8. 5. The rAAV of claim 1,wherein the expression control sequences comprise a promoter.
 6. TherAAV of claim 5, wherein the promoter is selected from a CB7 promoter, aTBG promoter, a Nkcc2 promoter, a uromodulin promoter, a Ksp-cadherinpromoter, and a THP gene promoter.
 7. The rAAV of claim 1, wherein theexpression control sequences comprise one or more of an intron, a Kozaksequence, a polyA, and post-transcriptional regulatory elements.
 8. TherAAV of claim 1, wherein the AAV capsid is an AAV8, an AAVrh64R1, anAAV9, an AAVhu.37, an AAVrh10 capsid, or a variant thereof.
 9. The rAAVof claim 1, wherein the capsid is an AAV8 capsid.
 10. A pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier and therAAV according to claim
 1. 11. A recombinant adeno-associated virus(rAAV) comprising an AAV capsid having packaged therein a vector genome,wherein said vector genome comprises a nucleotide sequence encoding afunctional canine EPO, inverted terminal repeat sequences, andexpression control sequences that direct expression of the EPO in a hostcell, wherein the nucleotide sequence encoding the canine EPO comprisesnucleotides 121 to 618 of the nucleotide sequence of SEQ ID NO: 7 andencodes at least amino acids 41 to 206 of the amino acid sequence of SEQID NO:
 3. 12. The rAAV of claim 11, wherein the EPO nucleotide sequenceencodes amino acids 1 to 206 of SEQ ID NO:
 3. 13. The rAAV of claim 12,wherein the nucleotide sequence encoding the canine EPO comprises SEQ IDNO:
 7. 14. The rAAV of claim 11, wherein the encoded canine EPO aminoacid sequence comprises the amino acids 41 to 206 of SEQ ID NO: 3 incombination with a heterologous leader sequence.
 15. The rAAV of claim11, wherein the expression control sequences comprise a promoter. 16.The rAAV of claim 15, wherein the promoter is selected from a CB7promoter, a TBG promoter, a Nkcc2 promoter, a uromodulin promoter, aKsp-cadherin promoter, and a THP gene promoter.
 17. The rAAV of claim11, wherein the expression control sequences comprise one or more of anintron, a Kozak sequence, a polyA, and post-transcriptional regulatoryelements.
 18. The rAAV of claim 11, wherein the AAV capsid is an AAV8,an AAVrh64R1, an AAV9, an AAVhu.37, an AAVrh10 capsid, or a variantthereof.
 19. The rAAV of claim 11, wherein the capsid is an AAV8 capsid.20. A pharmaceutical composition comprising a pharmaceuticallyacceptable carrier and the rAAV according to claim 11.